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Myosin light-chain kinase regulates endothelial calcium entry and endothelium-dependent vasodilation ¹

HIROSHI WATANABE^*2, QUANG-KIM TRAN^*, KAZUHIKO TAKEUCHI^*, MITSUHIRO FUKAO, MING Y. LIU, MORIO KANNO, TOSHIO HAYASHI, AKIHISA IGUCHI, MINORU SETO^§ and KYOICHI OHASHI^*

^* Department of Clinical Pharmacology and Therapeutics, Hamamatsu University School of Medicine, 3600 Handa-cho, Hamamatsu 431-3192;
Department of Pharmacology, Hokkaido University School of Medicine, Sapporo 060-8638;
Department of Geriatric Medicine, Nagoya University School of Medicine, Nagoya 466-8560; and
^§ Life Science Research Center, Asahi Chemical Industry Company, Ltd., Fuji 416-0934, Japan

²Correspondence: Hiroshi Watanabe, M.D., Ph.D., Department of Clinical Pharmacology and Therapeutics, Hamamatsu University School of Medicine, 3600 Handa-cho, Hamamatsu 431-3192, Japan. E-mail: hwat{at}hama-med.ac.jp

SPECIFIC AIMS

We used a combination of pharmacological and molecular approaches to consolidate the regulatory role of myosin light chain kinase (MLCK) in Ca²⁺ entry (CE) in endothelial cells and to clarify the relevant mechanism. Most importantly, we investigated the role of MLCK on Ca²⁺-dependent functions of endothelial cells (ECs), including production of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF).

PRINCIPAL FINDINGS

1. MLCK antisense attenuates BK- and TG-induced Ca²⁺ responses
To consolidate MLCK’s role in endothelial Ca²⁺ signaling, we evaluated Ca²⁺ responses to bradykinin (BK) and thapsigargin (TG) in ECs transfected with MLCK sense and antisense oligonucleotides. In Ca²⁺-containing medium, BK (10 nM) rapidly increased fura-2 ratio from 0.53 ± 0.13 (n=14) to a maximum of 4.34 ± 0.36 for 90 s, and maintained it at 2.92 ± 0.55, 5 min after application (Fig. 1A ). MLCK antisense significantly inhibited both the peak and plateau phase (peak: 2.64 ± 0.73; plateau: 1.17 ± 0.44; n=14). MLCK sense, however, had no effect. Likewise, in Ca²⁺-containing medium, TG (1 µM) gradually increased fura-2 ratio from 0.54 ± 0.15 (n=14) to 4.36 ± 0.46 (n=14) in 3 min, which was sustained at 4.20 ± 0.51, 10 min after TG application (Fig. 1B ). MLCK antisense substantially prevented both the peak and plateau phase (peak: 1.23 ± 0.27; plateau: 1.18 ± 0.53, n=14), and MLCK sense again had no effect.

View larger version (59K): [in this window] [in a new window]   Figure 1. MLC phosphorylation-independent control of endothelial Ca2+ signaling by MLCK. A) BK (10 nM)-stimulated Ca2+ response in ECs subjected to either control (CTL, open bars; n=14 from three separate experiments), transfection with MLCK antisense oligonucleotides (Antisense, closed bars; n=14), or MLCK sense oligonucleotides (Sense, shaded bars, n=14). Plateau Ca2+ response was evaluated 5 min after the addition of BK. *, P < 0.01 versus CTL. B) TG (1 µM)-stimulated Ca2+ response in ECs subjected to either control (CTL, open circles; n=14 from three separate experiments), transfection with MLCK antisense oligonucleotides (Antisense, closed circles; n=14) or transfection with MLCK sense oligonucleotides (Sense, closed triangles; n=14). TG-stimulated Ca2+ response was significantly attenuated in MLCK antisense ECs 1 min after the addition of TG (P<0.01). C) Immunostaining of MLC in cultured porcine aortic ECs revealed unphosphorylated (upper bands, MLC-UP, or open areas), monophosphorylated (middle bands, MLC-P, or shaded areas) and diphosphorylated MLC (lower bands, MLC-PP, or closed areas), respectively. The degree of phosphorylation is expressed as percentage of the total extracted MLC. In the presence of 1 mM extracellular Ca2+, TG (1 µM, 2 min) stimulated the formation of diphosphorylated MLC, which was inhibited by either transfection with MLCK antisense oligonucleotides (TG/AS) or treatment with 100 µM ML-9 (TG/ML9). D) In the presence of 1 mM extracellular Ca2+, BK (10 nM, 2 min) stimulated the formation of diphosphorylated MLC, which was abolished by the removal of extracellular Ca2+ (BK/ 0 Ca). Calyculin A (1 µM, 10 min) converted all MLC to diphosphorylated form (CA). Data were from three separate experiments.

2. MLCK regulates myosin light-chain (MLC) diphosphorylation through activation of CE
A key role of MLCK is to phosphorylate MLC. Western blotting revealed a significant increase in MLC diphosphorylation following stimulation with TG (from 0 % to 35.1 ± 5.4 %) (Fig. 1C ), which was inhibited significantly in MLCK antisense ECs (MLC-PP: 5.2 ± 3.8%). This diphosphorylation was abolished by the MLCK inhibitor ML-9 (MLC-PP: 0.0 ± 0.0%). These results indicate that MLCK activity was inhibited significantly by manipulations that inhibit endothelial CE. In Ca²⁺-containing medium, MLC diphosphorylation was increased significantly by 10 nM BK (MLC-PP: 31.2 ± 12.5%). However, MLC was not diphosphorylated by BK when the medium was Ca²⁺-free (Fig. 1D ). This finding means that MLC diphosphorylation is totally dependent on Ca²⁺ entry. In addition, calyculin A (1 µM), an inhibitor of type 1 and 2A phosphatases, converted all MLC to diphosphorylated form (MLC-PP: 100.0 ± 0.0%) (Fig. 1D ) but failed to increase fura-2 ratio (0.58 ± 0.13 vs. 0.61 ± 0.14, before and after calyculin A treatment, respectively; n=14).

3. MLCK regulates BK- and TG-induced endothelial NO production and acetylcholine (ACh)-induced hyperpolarization in SMCs
To examine the role of MLCK in endothelium-dependent vasodilation, we first tested the effects of MLCK antisense oligonucleotides and MLCK inhibitors on BK- and TG-stimulated NO production. Unstimulated ECs had low basal NO production of 0.57 ± 0.06 nmol /10⁶ cells. Treatment for 10 min with BK (10 nM) and TG (1 µM) augmented NO production to 3.56 ± 0.47 and 5.79 ± 0.88 nmol/10⁶ cells, respectively (Fig. 2A ). NO production stimulated by both BK and TG was reduced significantly in MLCK antisense cells, to 1.04 ± 0.08 and 1.27 ± 0.28 nmol/10⁶ cells, respectively. ML-9 (100 µM) and wortmannin (100 µM), a potent inhibitor of both MLCK and phosphatidylinositol 3 (PI₃)-kinase, almost abolished BK-and TG-stimulated NO production.

View larger version (23K): [in this window] [in a new window]   Figure 2. Regulation by MLCK of BK-and TG-stimulated NO production from ECs and ACh-induced hyperpolarization of SMCs. A) Treatment for 10 min with BK (10 nM) and TG (1 µM) greatly increased NO production from basal level (CTL), which was strongly inhibited by either transfection with MLCK antisense (BK/AS or TG/AS) or pretreatment with ML-9 (100 µM, 10 min) (BK/ML9 or TG/ML9) or wortmannin (100 µM, 30 min) (BK/Wort or TG/Wort), respectively. Data were from four separate experiments. *, P < 0.01 versus either BK in BK-treated group or TG in TG-treated group; #, P < 0.05; ##, P < 0.01 versus control. B) Dose-dependent inhibition by ML-9 of ACh-induced hyperpolarization of SMCs. Rat mesenteric arterires were pretreated for 10 min with the specified concentrations of ML-9 before application of ACh (1 µM). C) Time-dependent inhibition by wortmannin of ACh-induced SMC hyperpolarization. Rat mesenteric arteries were pre-incubated with wortmannin (100 µM) for the specified periods before application of ACh (1 µM).

We proceeded to examine MLCK’s role in EDHF production assessed by SMC membrane potential in rat mesenteric artery. Resting membrane potential was –51 ± 0.8 mV (mean ± SE; n=10), which was not affected by endothelial denudation (not shown). In endothelium-denuded tissues, ACh (1 µM) did not change membrane potential. In tissues with intact endothelium, however, ACh (1 µM) hyperpolarized the membrane by 13 ± 1.1 mV (n=10), which was maintained as long as the tissues were exposed to ACh. Neither ML-9 (100 µM) nor wortmannin (100 µM) affected the resting membrane potential. However, ML-9 inhibited ACh-induced hyperpolarization by 15% (11 ± 1.1 mV, n=5, p<0.05) and 62% (5.0 ± 0.7 mV, n=5, p<0.01) at 30 µM and 100 µM, respectively (Fig. 2B ). Wortmannin also dose- and time-dependently inhibited this hyperpolarization by 8%, 23%, and 38% at 10 µM, and 23%, 69%, and 100% at 100 µM, after pretreatment for 10, 20, and 30 min, respectively (Fig. 2C ) (n=5, p<0.01).

CONCLUSIONS

Agonist- and fluid flow-stimulation triggers endothelial CE, which stimulates production of NO, PGI₂, and EDHF and leads subsequently to vasodilation. Recently, we reported that inhibitors of MLCK inhibited agonist- and fluid flow-stimulated CE. Although these findings suggest that MLCK probably controls endothelial CE, further consolidation was necessary with methods other than the use of kinase inhibitors. The significant inhibition of CE and MLC diphosphorylation by MLCK antisense oligonucleotides in this study is solid evidence for the regulatory role of MLCK in endothelial CE.

We also previously showed that agonist-induced CE was well-correlated with MLC diphosphorylation, which was confirmed by the findings here. However, it was unclear whether MLCK activates CE by affecting directly the transmembranous CE pathways or indirectly through MLC phosphorylation. MLCK regulates many contractile events in both muscle and non-muscle cells, including ECs, and it could be that MLCK inhibition would prevent cytoskeletal reorganization surrounding the CE pathways and secondarily modulate CE. Alternatively, MLCK might also regulate CE independently of MLC diphosphorylation, acting directly on the CE pathways or eliciting production of another second messenger that activates them. We have now clarified that diphosphorylation of MLC does not stimulate CE and that MLC diphosphorylation is itself dependent on CE. It is likely, then, that MLCK regulates CE independently of MLC phosphorylation (Fig. 3A ) and that the level of diphosphorylated MLC may in this regard be only a marker of MLCK activity in ECs. These data strongly indicate that MLCK has another target to phosphorylate other than MLC in the activation of CE.

View larger version (21K): [in this window] [in a new window]   Figure 3. Schematic diagrams for the regulation by MLCK of endothelial CE and endothelium-dependent vasodilation. A) MLCK, activated following internal Ca2+ store depletion in ECs by agonists, shear stress, or endoplasmic reticulum Ca2+ ATPase inhibitors, triggers CE, which causes MLC diphosphorylation and stimulates production of NO and EDHF. B) Activation of MLCK in ECs causes vascular relaxation via increased [Ca2+]i, NO, and EDHF, thereby counterbalancing its well-known vasoconstrictor effects in smooth muscle cells.

Most importantly, we have shown for the first time that MLCK, via its action in ECs, strongly controls production and release of NO and EDHF. Endothelial NO production is known to correlate more with transmembranous CE than intracellular store Ca²⁺ release and thus there likely is causality between the inhibitory effects that MLCK inhibition showed on CE and NO production in this study. Production and release of proposed EDHF have been indicated to depend partly on [Ca²⁺]_i and thus the inhibition of ACh-induced hyperpolarization by ML-9, and wortmannin here is likely due to inhibition of the Ca²⁺ response. Although at 100 µM both agents blocked agonist-stimulated CE to the same extent, ML-9 only partially inhibited ACh-induced hyperpolarization, while wortmannin almost abolished this response. Because ACh-induced hyperpolarization is dependent on both Ca²⁺ release and CE, its incomplete inhibition by ML-9 could be attributed to the compound’s effect to inhibit only the influx portion of the ACh-induced Ca²⁺ response. ACh also activates PI₃-kinase, which stimulates IP₃ production and intracellular Ca²⁺ release. The fact that wortmannin inhibits both PI₃-kinase and MLCK may thus account for its complete inhibition of ACh-induced hyperpolarization.

Activation of MLCK in smooth muscle cells causes vasoconstriction. We have now established that MLCK controls endothelial CE and production of both NO and EDHF. It is likely that MLCK-activated CE stimulates NO and EDHF production and thus leads to vasodilation. We now know that MLCK possesses a counter-balancing role in vascular regulation: vasoconstriction via direct action on SMCs and vasodilation via action on ECs (Fig. 3B ).

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0587fje To cite this article, use (December 8, 2000) FASEB J. 10.1096/fj.00-0587fje

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